The present invention relates to novel uses of fibrin, especially plasma-derived fibrin. The invention includes the use of fibrin alone as an extracellular matrix. Further, the invention includes combining anticoagulated plasma, clotting agents and cells together to form engineered tissue. The engineered tissue can include the synthetic manufacture of specific organs or xe2x80x9corgan-likexe2x80x9d tissue.
Fibrin is a natural clotting agent. Therefore, fibrin and fibrin derivatives have commonly been used in hemostatic applications. Its use in analogous applications and in connection with various reinforcing additives has been known and well-documented.
The role of fibrin in the metastatic implantation and growth of cancer cells is also known. Cancer cells are typically highly thrombogenic. This causes rapid clot formation (fibrin formation) around cancer cells and, unfortunately for many cancer patients, very efficiently facilitates the growth of tumors.
In another field, tissue and organ replacement in patients with failing or damaged organ function is hampered by several significant problems. First, the source of replacement tissue or a replacement organ is typically a living related or cadaveric donor. Both of these sources are limited in number and carry the risk of exposing a recipient to pathologic viruses. Second, since the source of the replacement tissue or organ (with the exception of a living identical twin donor) is genetically distinct from the recipient, and the problems of organ rejection and graft versus host disease are significant. Both of these problems can be treated with immunosuppression, but this can cause significant side effects and dramatically increases the risk of infection in a patient.
In a still further field, the emerging techniques with respect to gene transfer can be dangerous when performed in vivo. In other words, in vivo gene transfer can expose a recipient to various complications associated with the processes used to transfer DNA and/or gene sequences into the target cell. Further, there are limitations to known gene therapies, for instance, with respect to engineering viral coats large enough to accept large genes such as the one for Factor VIII (anti-hemophilic factor).
In a still further field of study, the science of chemotherapy for cancer patients is, at least at some level, based on estimates of effectiveness of various treatments in combating a patient""s cancer cells. There is no efficient way to identify a patient""s cancer cells response to chemotherapy in vivo.
Accordingly, it is an object of the present invention to overcome the foregoing limitations by providing plasma-derived fibrin as an extracellular matrix. In the fields of tissue and organ replacement specifically, the use of fibrin, and specifically, a patient""s own cells and fibrin, could overcome problems of potential infection and rejection. In the field of gene transfer, the manipulation of cells could take place in vitro where cells that are cultured to be inserted into an extracellular matrix comprised of fibrin could be more easily manipulated and tested. Finally, in the context of chemotherapy, the plasma-derived fibrin extracellular matrix implanted with a patient""s tumor cells could be used to identify in vitro the susceptibility of those cells to alternative chemotherapy treatments. Each of the foregoing alternatives can result in safer and more predictable medical practices.
In one embodiment, there is provided an extracellular matrix for promoting cell growth comprising anticoagulated plasma and clotting agent The resulting fibrin can be formed of electrospun fibrin fibers or electroaerosol fibrin droplets. Alternatively, the fibrin may be formed of extruded fibrin or sheared fibrin.
In another embodiment, the invention includes an engineered tissue that includes a suspension comprising anticoagulated plasma, clotting agent and cells. The cells may be stem cells and/or committed stem cells. Further alternatively, the suspension may further comprise differentiation inducers, such as DNA sequences (e.g. Myo D to make muscle) or pharmaceuticals (e.g. retonic acid and others). Also, the engineered tissue may have a predetermined shape and the suspension of resulting fibrin and cells has substantially the same predetermined shape.
The invention further includes the method of forming an engineered tissue comprising mixing together anticoagulated plasma, a clotting agent and cells. Additionally, fibrinolytic inhibitors may be added at the time of mixture to prevent degradation of the resulting fibrin matrix before about two days or longer depending on the tissue formed.
In still a further embodiment, the invention includes a method of manufacturing an extracellular matrix. The method includes streaming an electrically-charged solution comprising fibrin onto a grounded target substrate under conditions effective to deposit the fibrin on said substrate to form an extracellular matrix. The electrically charged solution may further comprise fibrinogen and thrombin. Alternatively, the electrically charged solution may comprise plasma and thrombin. The fibrin streamed on to the substrate may comprise either electrospun fibers or electroaerosol droplets.
In still a further embodiment, the invention includes a method for manufacturing an extracellular matrix having a predetermined shape. The method includes preselecting a mold adapted to make the predetermined shape and filling the mold with a suspension comprising fibrin. The suspension may further comprise fibrinogen and thrombin. Alternatively, the method may comprise preselecting a mold adapted to make the predetermined shape wherein the mold comprises a grounded target substrate. Then, an electrically charged solution comprising fibrin is streamed onto the grounded target substrate under conditions effective to deposit the fibrin on the substrate to form the extracellular matrix having the predetermined shape. The fibrin streamed onto the substrate may comprise electrospun fibers or electroaerosol droplets.
In still a further embodiment, a method of manufacturing an engineered tissue comprising fibrin and cells comprises streaming an electrically charged solution comprising the fibrin and cells onto a grounded target substrate under conditions effective to deposit the fibrin and cells onto the substrate. The fibrin and cells streamed onto the substrate may comprise electrospun fibers or electroaerosol droplets. The cells may be stem cells and/or committed stem cells. Alternatively, they may be myoblast cells.
In still a further embodiment, a method is disclosed for manufacturing an extracellular matrix. The method includes an electrically grounded substrate and further providing a plurality of reservoirs containing polymer solutions. The reservoirs are connected substantially at a single orifice that allows the mixture of solutions from the reservoirs upon exit from the reservoirs. The solutions are electrically charged and the mixture of solutions is streamed onto the substrate to form an extracellular matrix. In an alternative embodiment, the plurality of reservoirs comprises first and second reservoirs. The first reservoir has a solution comprising fibrinogen and the second reservoir has a solution comprising thrombin.
In still a further embodiment, the invention includes a method for testing the effectiveness of cancer therapy treatments in vitro. The method includes manufacturing engineered tissue comprising anticoagulated plasma, clotting agent and cancer cells. It further includes preparing a plurality of samples of the engineered tissue and subjecting a plurality of cancer therapy treatments to the samples of engineered tissue. It further includes evaluating the relative effectiveness of the cancer therapy treatments. This method may alternatively include manufacturing engineered tissue by streaming an electrically charged solution comprising fibrin and cancer cells on to a grounded target substrate under conditions effective to deposit the fibrin and cancer cells onto the substrate. Also, the cancer cells may be obtained from a patient who is in need of cancer therapy treatments.